Adult pancreatic stem and progenitor cells may serve as an alternative and novel source of insulin-producing beta cells for cell replacement therapy in patients with type 1 diabetes. However, the existence of these cells is highly controversial. Our laboratory was the first to establish in vitro 3-dimensional culture systems which allow the quantification of putative adult pancreatic stem and progenitor cells that give rise to colonies (also known as organoids). Using colony assays, we discovered that there are rare cells, ~1% among total dissociated cells in the adult murine pancreas, that are capable of self-renewing long-term, and differentiating into three major pancreatic lineages: the acinar, ductal and endocrine cells. We have named these rare cells ?pancreatic colony-forming units (PCFUs)?. Self-renewal and differentiation are two criteria necessary to define a stem cells, and therefore our results suggest that PCFUs are the putative stem and progenitor cells in the adult pancreas. Currently, there are three pressing issues facing the field of adult pancreatic stem cell biology: 1) adult stem cells being a minority of cells, 2) no appropriate in vivo injury models to study them, and 3) no information on gene signatures that allow specific identification of pancreatic stem cells across species and development. To address these issues, the primary goals of this proposal are to 1) fractionate pancreas based mostly on cell surface markers, 2) sort/enrich/purify PCFUs from pancreases of adult mice, adult humans and human embryos, and 3) identify a gene signature of pancreas stem cells. The hypothesis is that stem cells exist in the adult pancreas that express developmentally- and evolutionally-conserved genes. The secondary goal is to determine which injury models will activate adult pancreatic stem cells in vivo. Aim 1 will focus on adult murine PCFUs. We will characterize an already highly-enriched population of cells identified from the preliminary study using fluorescence-activated cell sorting and in vitro colony assays, and determine injury conditions that will activate these murine PCFUs after transplantation in vivo. Aim 2 will focus on adult human PCFUs. We will identify cell surface markers and determine whether adult human PCFUs can be enriched and expanded in a GMP-compatible suspension culture, guided by our in vitro human colony assay. Aim 3 will focus on human fetal PCFUs. Preliminary data showed that human fetal PCFUs can also form colonies in the colony assay originally designed for adult human PCFUs. We will therefore use similar strategy to enrich and characterize human fetal PCFUs, and use the resulting knowledge to serve as a counterpoint to compare to adult human PCFUs and identify unique gene signatures/markers. A successful completion of the proposed research will have major impacts on 1) the existence of adult human pancreatic stem and progenitor cells, 2) identification of novel markers for adult pancreatic stem cells, and 3) providing a potentially safer adult cell source, compared to the pluripotent stem cells that have potential to form teratoma, for cell replacement therapy of type 1 diabetes.